Flow-induced K secretion (FIKS) in the cortical collecting duct (CCD) is mediated by the iberiotoxin (IbTX)-sensitive Ca2+/stretch-activated BK channel, comprised of pore-forming 1 and accessory 2 subunits. The channel is detected in both Na absorbing principal cells (PC) and acid-base transporting intercalated cells (ICs). We hypothesize that the BK channel is localized in a macromolecular complex, comprised of mechanosensitive apical Ca2+ channels and a variety of kinases/phosphatases as well as other signaling molecules, anchored to the cytoskeleton, and that an increase in tubular fluid flow rate leads to IC- and PC-specific responses determined, in large part, by the cell-specific composition of the BK channels therein. This hypothesis will be tested in two Specific Aims (SAs). SA1 proposes to identify the role of NKCC1 in FIKS in the CCD. As the density of BK channels in IC exceeds that in PC, the IC would be the logical candidate to mediate FIKS. However, this role would require that ICs have a robust mechanism to sustain a high steady-state [K]i. We propose that K uptake in ICs is mediated by a basolateral Na-K-2CI cotransporter (NKCC1). To test this, we will first (A) examine whether basolateral bumetanide (NKCC inhibitor) and luminal IBX inhibit the same transport pathway in in vitro microperfused CCDs; (B) test whether functional NKCC is present along the basolateral membranes of IC and/or PC using fluorescent functional probes; and (C) examine the capacity of CDs from mice with targeted deletion of NKCC1 for FIKS. SA2 will test whether BK channels in PC and IC are differentially regulated by mechano-activated signaling pathways due to the expression of unique cell-specific BK1 variants and 2 isoforms. To this end, we will (A) determine the molecular composition of BK channels in individual IC and PC, and examine whether variant and isoform expression is regulated by dietary K intake and fluid shear stress (FSS), and then examine the roles of (B) NO/cGMP/PKG, (C) MAPK, (D) palmitoylation and (E) FSS induced phosphorylation in the regulation of BK channels in the CCD. We anticipate that the proposed studies will uncover mechanisms involved in the development/maintenance of disorders of urinary K excretion and identify potential targets for therapies to treat K imbalances. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to examine the molecular mechanisms underlying regulation of BK potassium channels in the distal nephron of the kidney. The studies will define how these channels are suppressed under conditions of low urinary flow rates, and activated to secrete potassium into the urine when urinary flow rates increase. This work has the potential to uncover mechanisms involved in the development and/or maintenance of disorders of potassium excretion and identify potential targets for novel therapies to treat imbalances in potassium balance.